Resveratrol inhibits phenotypic and functional maturation of murine bone marrow-derived dendritic cells

This study investigated the effects of resveratrol, a natural polyphenol found in grapes and grape products such as wine and having a wide range of biological and pharmacological activities effecting on the phenotypic and functional maturation of bone marrow (BM)-derived dendritic cells (DC). Resveratrol inhibited the expression of costimulatory molecules (CD80 and CD86), and major histocompatibility complex (MHC) classes I and II significantly, and had the same effect dose-dependently on DC. Resveratrol also significantly suppressed the ability of BM-DC to produce intracellular IL-12 p40/p70 and secretory IL-12 p70 in response to lipopolysaccharides (LPS) stimulation. Resveratrol-treated DC were highly efficient in antigen capture via mannose receptor-mediated endocytosis. Also, they were poor stimulators of na?ve allogeneic T-cell proliferation and induced lower levels of IL-2 in responding T cells. These results indicate the immunosuppressive properties of resveratrol, which may be therapeutically useful in controlling chronic immune and/or inflammatory diseases through the down-regulation of DC differentiation and maturation.     

Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells

Ginkgetin, a biflavone from Ginkgo biloba leaves, was previously reported to be a phospholipase A(2) inhibitor and this compound showed the potent antiarthritic activity in rat adjuvant-induced arthritis as well as analgesic activity. This investigation was carried out to find effects on cyclooxygenase-2 (COX-2) in vitro effect. Ginkgetin inhibits COX-2 dependent phases of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC(50) values of 0.75 microM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD(2) product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C(4) (LTC(4)) in a dose dependent manner, with an IC(50) value of 0.33 microM. These results demonstrate that ginkgetin has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound also inhibited degranulation reaction in a dose dependent manner, with an IC(50) value of 6.52 microM. Therefore, this compound might provide a basis for novel anti-inflammatory agents.     

Naturally occurring biflavonoid, ochanflavone, inhibits cyclo-oxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells

Ochnaflavone is a medicinal herbal product isolated from Lonicera japonica that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC50 values of 0.6 microM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD2 product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 6.56 microM. These results demonstrate that ochnaflavone has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound strongly inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 3.01 microM. Therefore, this compound might provide a basis for novel anti-inflammatory drugs.     

Regulation of proliferation of mouse bone marrow-derived mast cells by activated fibroblasts

Nitric oxide (NO) is synthesized by various cells involved in inflammatory reactions and may then act on mast cells. In the present work, we attempted to clarify the role of this molecule on the proliferation of mouse bone marrow derived-mast cells (BMMC). Swiss 3T3 fibroblasts produced nitrite (NO₂) and nitrate (NO₃) upon treatment with interferon γ (IFN-γ). This formation was dependent ofL-arginine and could be inhibited by theL-arginine analogue N^G-monomethyl-L-arginine (N ^GMMA). The effect of IFN-γ was drastically increased by cotreatment with tumor necrosis factor γ (TNF-γ). BMMC were maintained in vitro for as long as 30 days when cocultured with Swiss 3T3 fibroblasts. Coculture withN ^GMMA, significantly increased the number of BMMC. These results indicate that NO involves the inhibition of proliferation of BMMC when cocultured with Swiss 3T3 fibroblasts.     

Inhibitory effects of Piper betle on production of allergic mediators by bone marrow-derived mast cells and lung epithelial cells

The leaves of the Piper betle Linn. (Piperaceae) are used in traditional medicine and possess anti-oxidant, anti-bacterial, anti-fungal, anti-diabetic and radioprotective activities. However, little is known about their anti-allergic activity. Therefore, the effects of P. betle ethanolic extract (PE) on the production of histamine and granulocyte macrophage-colony-stimulating factor (GM-CSF) by murine bone marrow mast cells (BMMCs) and on the secretion of eotaxin and IL-8 by the human lung epithelial cell line, BEAS-2B, were investigated in vitro. PE significantly decreased histamine and GM-CSF produced by an IgE-mediated hypersensitive reaction, and inhibited eotaxin and IL-8 secretion in a TNF-alpha and IL-4-induced allergic reaction. The results suggest that P. betle may offer a new therapeutic approach for the control of allergic diseases through inhibition of production of allergic mediators.     

Salmeterol inhibits anaphylactic histamine release from guinea-pig isolated mast cells

Abstract— Salmeterol (1 Nm-100μm) showed an inhibitory action on anaphylactic histamine release from mast cells, isolated from pleural and peritoneal cavities of actively sensitized guinea-pigs and stimulated by incubation with allergen. The effect is concentration-dependent and is reduced by the β-adrenoceptor antagonist propranolol (1 μm). This study supports the hypothesis of an antiinflammatory property of salmeterol, which concerns cells involved in the early phases of asthma.     

Dual inhibition of cyclooxygenases-2 and 5-lipoxygenase by deoxypodophyllotoxin in mouse bone marrow-derived mast cells

Deoxypodophyllotoxin (Anthricin) is a medicinal herbal product isolated from Anthriscus sylvestris HOFFM. that inhibits cyclooxygenase-2 (COX-2) and COX-1-dependent phases of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC(50) values of 1.89 microM and 65.3 microM, respectively. This study also found that this compound inhibited COX-1 and 2-dependent conversion of the exogenous arachidonic acid to PGD(2) in a dose-dependent manner with an IC(50) values of 0.01 microM and 12.1 microM, respectively using a COX enzyme assay kit. However, this compound did not inhibit COX-2 protein expression up to a concentration of 30 microM in the BMMC, indicating that deoxypodophyllotoxin directly inhibits COX-2 activity. Furthermore, this compound consistently inhibited the production of leukotriene C(4) (LTC(4)) in a dose dependent manner, with an IC(50) value of 0.37 microM. These results demonstrate that deoxypodophyllotoxin has a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitory activity, and therefore this compound might provide a basis for novel anti-inflammatory drugs.     

Forskolin inhibits the release of histamine from human basophils and mast cells

We found that forskolin (10(-7) to 3 X 10(-5) M) caused dose-related inhibition of antigen-induced histamine release from human basophil leukocytes. The dose-response inhibition curve was paralleled by a forskolin-induced increase in cyclic AMP (cAMP) levels in human leukocyte preparations. The kinetics of inhibition of histamine release and of the increase in leukocyte cAMP were the same. In a second series of experiments we evaluated the effect of forskolin on antigen-induced histamine release from chopped human lung passively sensitized with serum from an allergic patient. Forskolin (10(-7) to 3 X 10(-5) M) dose-dependently inhibited the release of histamine from human lung mast cells. Thus forskolin appears to modulate the release of mediators of the immediate hypersensitivity reaction, presumably through activation of adenylate cyclase in human basophils and mast cells.     

5-Azacytidine inhibits proliferation and induces apoptosis of mouse bone marrow-derived mesenchymal stem cells

Abstract

In this study, we investigated the effect of 5-azacytidine on proliferation and apoptosis of bmMSCs. After exposure to different concentrations of 5-azacytindine, bmMSC proliferation and apoptosis were measured by MTT assay, Caspase-3 staining and western blotting. Our results show that 1?μM 5-azacytidine has no significant effect on viability of bmMSCs; however, 5–20?μM 5-azacytindine significantly inhibits bmMSC proliferation and expression of phospho-Akt; 10 and 20?μM 5-azacytidine markedly increases bmMSC apoptosis and expression of Caspase-3, Annexin V and Bax, and decreases expression of Bcl2. Our findings indicate that 5-azacytidine at the commonly used doses has toxicity to bmMSCs.     

Ketotifen inhibits paf-acether biosynthesis and beta-hexosaminidase release in mouse mast cells stimulated with antigen

Acetyl-CoA acetyltransferase (1-O-alkyl-sn-glycero-3-phosphocholine) is a key enzyme in paf-acether biosynthesis. Its immunological activation as related to paf-acether formation was investigated in mast cells derived from mouse bone marrow. The action of ketotifen, a prophylactic anti-asthma drug, on the antigen-induced activation of acetyltransferase and on the release of paf-acether and beta-hexosaminidase was studied in mast cells. Mast cells were sensitized with dinitrophenyl-specific monoclonal IgE and preincubated for 15 min at 37 degrees C with various concentrations of ketotifen or vehicle prior to challenge with dinitrophenyl coupled to bovine serum albumin (40 ng/ml). Acetyltransferase activity and mediator formation and release were measured. Ketotifen inhibited dose dependently the antigen-induced paf-acether formation and release, beta-hexosaminidase release and acetyltransferase stimulation. The IC50 values were 20.0 +/- 4.4, 11.8 +/- 6.2, 8.8 +/- 3.8 and 20.5 +/- 3.4 microM (mean +/- S.E.M., n = 3) respectively. Mast cells were preincubated with 50 microM ketotifen for 15 min at 37 degrees C then washed prior to antigen challenge. The release of paf-acether and beta-hexosaminidase and the stimulation of acetyltransferase were inhibited by 90.0 +/- 15.0, 91.0 +/- 15.0 and 88.0 +/- 11.0% (n = 3) respectively. In addition, Ca2+ entry was inhibited by 100% as assessed from Quin-2 fluorescence. Thus, the release of a preformed granular enzyme beta-hexosaminidase is inhibited by ketotifen together with the enzymatic formation of a newly formed mediator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine release from rat mast cells sensitized with mouse antiserum

The characteristics of the antigen-induced and non-antigen-induced histamine release from rat peritoneal mast cells sensitized in vitro with mouse anti-ovalbumin serum were investigated. The effects of some antiallergic drugs on these release reactions were also studied. Besides antigen-specific IgE antibody, heat-labile factor(s) responsible for the non-antigen-induced histamine release were found in mouse antiserum. Such factors were also present in normal mouse serum. In the absence of antigen, the combination of phosphatidyl serine and Ca++ induced some extent of histamine release from mast cells treated with these factors. From the present results it is suggested that quercetin selectively and verapamil primarily act to block calcium-gate opening resulting from antigen-antibody interaction on the mast cell membrane, while theophylline and disodium cromoglycate selectively inhibit the passage of calcium through open calcium channels.     

On the mechanism of histamine release from sodium fluoride-activated mouse mast cells

We have found that sodium fluoride-induced histamine release from mouse mast cells occurs in two separate steps, activation in the presence of fluoride and absence of calcium, and secretion triggered by calcium. The amount of released histamine was dependent either on the time of cell exposure to fluoride or on the final fluoride concentration in the incubation medium. The secretory step depends on the concentration of extracellular calcium; it increased as the concentration of calcium was increased. However, a substantial part of the release was both calcium and energy independent. This part, probably cytotoxic, increased markedly at the highest concentration and with extended times of cell exposure to fluoride. Among other divalent cations tested only strontium could partly substitute calcium to trigger secretion. The activating action of fluoride slowly decayed with time and addition of calcium for up to 2 h caused histamine release. Both steps were dependent on temperature and pH and were inhibited by antimycin A, suggesting that the reaction was enzymatic. The action of fluoride on mouse mast cells closely resembles its action on rat mast cells; however, some differences were also observed.     

Prostaglandin D2 inhibits IgE-mediated scratching by suppressing histamine release from mast cells

Effects of prostaglandin (PG) D(2), PGE(2), and PGI(2) on itch-associated scratching responses of mice and histamine release from the rat basophilic leukemia cell line RBL-2H3 were examined. PGD(2) and ketotifen but not PGE(2) and PGI(2) suppressed the scratching caused by ovalbumin injected into ovalbumin-sensitized mice. Ketotifen also suppressed compound 48/80-induced scratching but not PGD(2), PGE(2), and PGI(2). In vitro, PGD(2) suppressed the antigen-induced histamine release from RBL-2H3 cells, but PGE(2) and PGI(2) did not. These findings suggest that PGD(2) specifically suppressed IgE-mediated scratching by inhibiting IgE-mediated histamine release from mast cells.     

Super-oxidized solution inhibits IgE-antigen-induced degranulation and cytokine release in mast cells

Activation of the high affinity IgE receptor (Fc epsilonRI) through IgE-antigen complexes induces mast cell degranulation, synthesis of lipid mediators and cytokine production. These effects are involved in Type I hypersensitivity reactions and controlling them has been the main objective of many anti-allergic therapies. Here we report that pretreatment of murine bone marrow derived mast cells (BMMC) with super-oxidized solution (SOS) inhibits Fc epsilonRI dependent-beta hexosaminidase and cytokine release. This effect is exerted without altering total protein tyrosine phosphorylation, MAPK activation, cytokine mRNA accumulation or calcium mobilization after Fc epsilonRI triggering. Our data suggest that this neutral pH-SOS acts like a mast cell-membrane stabilizer inhibiting the cell machinery for granule secretion without altering the signal transduction pathways induced by IgE-antigen receptor crosslinking.     

成人骨髓间充质干细胞体外向视网膜细胞的诱导分化

目的研究取材于成人骨髓的间充质干细胞在一定诱导条件下向视网膜神经细胞的分化。方法成人骨髓经密度梯度离心得到的细胞,根据其高黏附特性体外培养获得间充质干细胞。利用流式细胞仪分析其细胞表型,在体外诱导使其向视网膜神经细胞分化并用免疫荧光法进行鉴定。结果从骨髓中分离培养的细胞具有成纤维细胞样形态,贴壁生长,表型相对均一,表面标志为CD90、CD44、CD147阳性;而CD34、CD38、CD45、CD14、HLA-DR阴性。体外诱导后可以得到表达nestin(神经干细胞标志物)、GFAP(神经胶质细胞标志物)和Rhodopsin(视网膜光感受器细胞标志物)阳性的细胞。结论从人骨髓中分离培养得到的间充质干细胞具有向视网膜神经细胞分化的潜能。     

Alpha-difluoromethylornithine inhibits bone resorption in vitro without decreasing beta-glucuronidase release

Our previous studies suggested that the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) inhibits bone resorption by mechanisms that are independent of polyamine depletion. To determine whether DFMO prevents calcitriol-stimulated bone resorption by acting at a step before or after osteoclast activation, we compared the effects of DFMO on release of calcium and beta-glucuronidase from cultured neonatal mouse calvaria. DFMO, at concentrations of 7.5-20 mM, inhibited release of calcium from calcitriol-stimulated calvaria but failed to inhibit the calcitriol-stimulated increase in beta-glucuronidase secretion. In contrast, ornithine, putrescine, spermidine, and spermine, at concentrations with effects on resorption comparable to those of DFMO, inhibited the effects of calcitriol on both calcium and beta-glucuronidase release. NaF (0.2 mM), like DFMO, inhibited calcitriol-stimulated calcium release without affecting medium beta-glucuronidase activity, whereas elevated phosphate (3 mM) inhibited both activities. The results suggest that DFMO, over the concentration range studied, inhibits calcium release by making the matrix resistant to resorption rather than by acting at a cellular locus.     

Resveratrol improves the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in rats with severe acute pancreatitis

ObjectiveBone marrow-derived mesenchymal stem cells (BMSCs) are effective in the treatment of severe acute pancreatitis (SAP), but their therapeutic effects could still be improved. In order to optimize the clinical application of BMSCs, we adopted the strategy of resveratrol (Res) pretreatment of BMSCs (Res-BMSCs) and applied it to a rat model of sodium taurocholate (NaT)-induced acute pancreatitis.MethodsSAP was induced by injection of 3% NaT into the pancreatic duct and successful induction of SAP occurred after 12 h. Rats were treated with BMSCs, Res or BMSCs primed with Res at 40 mmol/L, Vandetanib (ZD6474) daily oral dosages of 50 mg/kg vandetanib.ResultsRes stimulated BMSCs to secrete vascular endothelial growth factor A (VEGFA), activated the downstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, and inhibited pancreatic cell apoptosis. In addition, conditioned medium (CM) from Res-BMSCs enhanced the proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, increased resistance to apoptosis and promoted the expression of angiogenesis-related proteins CD31, VEGF and VEGFR2 in pancreatic tissue, but Vandetanib partly abolished these effects by blocking the VEGFA- mediated pathway.ConclusionResveratrol-preprocessed BMSCs can activate the PI3K/AKT signaling pathway in pancreatic cells and HUVECs through paracrine release of VEGFA; thus, achieving the therapeutic effect of resisting apoptosis of pancreatic cells and promoting regeneration of damaged blood vessels. Res pretreatment may be a new strategy to improve the therapeutic effect of BMSCs on SAP.     

Effects of methyl gallate on arachidonic acid metabolizing enzymes: Cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells

Methyl gallate (MG) is a medicinal herbal product that is isolated from Paeonia lactiflora that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with an IC50 values of 17.0 microM. This compound also found inhibited the COX-2-dependent conversion of the exogenous arachidonic acid to PGD2 in a dose-dependent manner with an IC50 values of 19.0 microM, using a COX enzyme assay kit. However, at concentrations up to 80 microM, MG did not inhibit COX-2 protein expression in BMMC, indicating that MG inhibits COX-2 activity directly. Furthermore, MG consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 5.3 microM. These results demonstrate that MG has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity, which might provide the basis for novel anti-inflammatory drugs.